Find the Targets and Answers that Matter

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1 Find the Targets and Answers that Matter Introducing the and Q Exactive Orbitrap MS for Targeted Lipid and Metabolic Profiling 1

2 1. Background Information 2. - Metabolite Coverage - Requirements - Kit Components - Kit Workflow 3. Analytical Performance 4. Biological Relevance & Applications 2

3 1. Background Information 2. - Metabolite Coverage - Requirements - Kit Components - Kit Workflow 3. Analytical Performance 4. Biological Relevance & Applications 3

4 Biocrates Life Sciences Company Profile Globally operating biotech company based in Innsbruck, Austria > 15 years of experience, with > 8 publications Fully integrated mass spectrometry based solutions for targeted metabolic phenotyping Services & kits: quantitative analysis of > 8 endogenous metabolites, statistical analysis, and biological interpretation Hypothesis driven biomarker research in pre-clinical, clinical, and epidemiological research 4

Targeted Metabolomics Kits for Standardized

Metabolites 188 2 17 AbsoluteIDQ p18 Kit -

acylcarnitines, hexoses - glycerophospho-

- primary bile acids - secondary bile acids

steroids *For US/Canada: License pending

5 Targeted Metabolomics Kits for Standardized & Quantitative Metabolic Phenotyping Metabolites AbsoluteIDQ p18 Kit - amino acids, biogenic amines - acylcarnitines, hexoses - glycerophospho- and sphingolipids Biocrates Bile Acids Kit - primary bile acids - secondary bile acids - human and rodent specific bile acids AbsoluteIDQ Stero17 Kit* - mineralocorticoids - glucocorticoids - sex steroids *For US/Canada: License pending QQQ MS lipids! HRAM MS Reprinted with permission of Thermo Fisher Scientific, CCL212 5

6 1. Background Information 2. - Metabolite Coverage - Requirements - Kit Components - Kit Workflow 3. Analytical Performance 4. Biological Relevance & Applications 6

Coverage Some interesting metrics HMDB metabolites: 39, Chemicals with 15 N, 13 C, 2 H labeled standards: 4 Typical number of features in discovery untargeted platforms:

7 Coverage Some interesting metrics HMDB metabolites: 39, Chemicals with 15 N, 13 C, 2 H labeled standards: 4 Typical number of features in discovery untargeted platforms: 1, Compounds Identified: 2? Source: David Wishart, presentation at MSACL 217 EU (Salzburg, Austria) September 217 Biocrates p4hr identified and quantifiable metabolites: 48 7

Metabolic and Lipid Profiling 48 Endogenous Metabolites 11 metabolite classes

amines (21) - Hexoses (1) Polar lipids - Phosphatidylcholines (172) -

lipids - Acylcarnitines (55) - Diglycerides (18) - Triglycerides (42) -

Spectrometer (+ UHPLC) Q Exactive Focus Q Exactive Q Exactive Plus Q Exactive HF

a Glance Ready-to-use quantitative kit for Q Exactive platforms High-throughput

8 Metabolic and Lipid Profiling 48 Endogenous Metabolites 11 metabolite classes 365 lipids & 43 small molecules Small Molecules - Amino acids (21) - Biogenic amines (21) - Hexoses (1) Polar lipids - Phosphatidylcholines (172) - Lysophosphatidylcholines (24) - Sphingomyelins (31) - Ceramides (9) Neutral lipids - Acylcarnitines (55) - Diglycerides (18) - Triglycerides (42) - Cholesteryl esters (14) Standard Thermo Scientific Orbitrap HRAM Mass Spectrometer (+ UHPLC) Q Exactive Focus Q Exactive Q Exactive Plus Q Exactive HF Reprinted with permission from Thermo Fisher Scientific, CCL212 Key Benefits at a Glance Ready-to-use quantitative kit for Q Exactive platforms High-throughput & minimal sample volume Simple & efficient automated workflow Robust platform for longitudinal & inter-lab comparability 8

9 Easy & Streamlined Workflow 9

Unique Standardized & Quality Controlled Kit Components Reagents & Consumables: Patented 96-well filter plates Internal standards Calibration standards Quality controls Instrument suitability test

10 Unique Standardized & Quality Controlled Kit Components Reagents & Consumables: Patented 96-well filter plates Internal standards Calibration standards Quality controls Instrument suitability test mix Methods & Protocols: Sample preparation protocol Instrument-specific acquisition & quantitation methods Workflow Manager MetIDQ Process guidance Automated technical validation All in one box Ready-to-use & user-friendly Standardized & quality controlled Only 1 µl sample required Validated for human plasma 1

11 Workflow Manager MetIDQ : Automated & Guided Process Transparent, Automated Workflow 1. Information Management Sample registration, plate layout design Worklist generation 11

Workflow Manager MetIDQ : Automated & Guided Process Transparent, Automated Workflow 1. Information Management Sample registration, plate layout design Worklist generation 2.

12 Workflow Manager MetIDQ : Automated & Guided Process Transparent, Automated Workflow 1. Information Management Sample registration, plate layout design Worklist generation 2. Quantification & Data Processing Automated signal identification & quantification Data merging 3. Automated Technical Validation Transparent display of generated data Analytical approval of the plate Inter-plate monitoring of QC results and precision 4. Data Evaluation and Export Data normalization (optional) Export of results in various file formats 5. Statistical Analysis (optional) Basic statistics & data visualization 12

13 Workflow Manager MetIDQ : Automated & Guided Process Transparent, Automated Workflow 1. Information Management Sample registration, plate layout design Worklist generation 2. Quantification & Data Processing Automated signal identification & quantification Data merging 3. Automated Technical Validation Transparent display of generated data Analytical approval of the plate Inter-plate monitoring of QC results and precision MetIDQ Software Transparent, automated & guided process Provides quantitative results 4. Data Evaluation and Export Data normalization (optional) Export of results in various file formats 5. Statistical Analysis (optional) Basic statistics & data visualization Reduced complexity & process safety Efficient & time saving High confidence in results 13

14 1. Background Information 2. - Metabolite Coverage - Requirements - Kit Components - Kit Workflow 3. Analytical Performance 4. Biological Relevance & Applications 14

Proven Excellent Accuracy The field MUST become more quantitative if findings are to be translated to

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jpg/@@images/2b4df4dc- 9f1a-4e5-a5f4-c2ce9a93fa8.

15 Proven Excellent Accuracy The field MUST become more quantitative if findings are to be translated to practical, clinical applications. David Wishart (215) MvMDYvMTBfMzJfMDBfNjE2X3dpc2hhcnQuanBnIl1d/wishart.jpg NIST SRM 195 Accuracy [%] Accuracy Range 9-114% n = 3 ages/griffin.jpg/@@images/2b4df4dc- 9f1a-4e5-a5f4-c2ce9a93fa8.jpeg If we could get over this barrier [to not quantitate our data] then it will make exchanging data between labs and studies so much easier, and we could really move forward as a community. Jules Griffin (MetaboNEWS, Sept 217) 15

16 Analytical Performance: Inter-day Comparability FIA-MS LC-MS B:\Biocrates\...\ _KIT3_SST_4 1/17/17 8:43:13 p15 testmix in RT: Relative Abundance Time (min) NL: 6.94E8 TIC MS _KIT3_SST_4 NL: 2.9E7 m/z= F: FTMS + c ESI Full ms [ ] MS _KIT3_SST_4 NL: 3.41E6 m/z= F: FTMS + c ESI Full ms [ ] MS _KIT3_SST_4 NL: 3.41E6 m/z= F: FTMS + c ESI Full ms [ ] MS _KIT3_SST_4 NL: 4.55E7 m/z= F: FTMS + c ESI Full ms [ ] MS _KIT3_SST_4 NL: 2.27E7 m/z= F: FTMS + c ESI Full ms [ ] MS _KIT3_SST_4 NL: 8.9E7 m/z= F: FTMS + c ESI Full ms [ ] MS _KIT3_SST_4 NL: 8.33E7 m/z= F: FTMS + c ESI Full ms [ ] MS _KIT3_SST_ _KIT2_QEF_Col25_SST_4 1/16/17 9:55:24 Testmix RT: Time (min) 5.49 NL: 7.9E9 TIC MS _KIT2_QEF_Col25_SST_4 NL: 1.24E9 m/z= F: FTMS + c ESI Full ms [55.-8.] MS _KIT2_QEF_Col25_SST_4 NL: 9.24E6 m/z= F: FTMS + c ESI Full ms [55.-8.] MS _KIT2_QEF_Col25_SST_4 NL: 1.67E8 m/z= F: FTMS + c ESI Full ms [55.-8.] MS _KIT2_QEF_Col25_SST_4 NL: 1.14E8 m/z= F: FTMS + c ESI Full ms [55.-8.] MS _KIT2_QEF_Col25_SST_4 NL: 1.74E8 m/z= F: FTMS + c ESI Full ms [55.-8.] MS _KIT2_QEF_Col25_SST_4 NL: 4.43E6 m/z= F: FTMS + c ESI Full ms [55.-8.] MS _KIT2_QEF_Col25_SST_4 NL: 5.96E2 Pump_Pressure A/D card _KIT2_QEF_Col25_SST_4 Accuracy 81-11%, Precision (CV) 1-25% Accuracy 92-14%, Precision (CV) 4-15% Inter-day comparability 16

Outstanding Inter-Lab Comparability LC-MS Part FIA-MS Part 3 labs, 5 instruments (Q Exactive

Focus) Reference lab 1 (Q Exactive Focus) 6 individual human plasma samples 6 individual human

Focus) 6k (Q Exactive HF) Accuracy 81-11%, Precision (CV) 1-25% Resolution for FIA-MS: 7k (Q

17 Outstanding Inter-Lab Comparability LC-MS Part FIA-MS Part 3 labs, 5 instruments (Q Exactive Focus & HF) 3 labs, 6 instruments (Q Exactive Focus, HF & Plus) Reference lab 1 (Q Exactive Focus) Reference lab 1 (Q Exactive Focus) 6 individual human plasma samples 6 individual human plasma samples Accuracy 92-14%, Precision (CV) 4-15% Resolution for LC-MS: 7k (Q Exactive Focus) 6k (Q Exactive HF) Accuracy 81-11%, Precision (CV) 1-25% Resolution for FIA-MS: 7k (Q Exactive Focus) 12k (Q Exactive HF) 7k (Q Exactive Plus) LC-MS based metabolites FIA-MS based metabolites 17

Excellent Analytical Performance Beger et al.

Worldwide Unique Ring Trials AbsoluteIDQ p18 Kit 6 laboratories, 3 instruments 7.

18 Excellent Analytical Performance Beger et al., Metabolomics (216) 12: 149 [ ] Biocrates commercially supplies kits for analysis of targeted areas of metabolism. [ ] Each kit provides advantages in interlaboratory use [ ]. Worldwide Unique Ring Trials AbsoluteIDQ p18 Kit 6 laboratories, 3 instruments 7.6% CV Siskos et al. (217), Anal Chem, 89: 1 Biocrates Bile Acids Kit 12 laboratories, 4 instruments 8.3% CV Pham et al. (216), J Appl Lab Med, DOI: jalm First ring trial scheduled for 18

19 1. Background Information 2. - Metabolite Coverage - Requirements - Kit Components - Kit Workflow 3. Analytical Performance 4. Biological Relevance & Applications 19

Mitochondrial functions - Mitochondrial beta oxidation of

20 Unique Analyte Coverage Provides Rich Information Biological Functions Membrane composition Cellular signaling Mitochondrial functions - Mitochondrial beta oxidation of fatty acids - Bioenergetic processes Oncology Cardiology Neurology Aging Diabetes 2

21 Multiple Rate-Limiting Enzymes Covered Rate-Limiting Step Slowest step of an enzymatic reaction that determines the rate (speed) at which the overall reaction proceeds. PLA2-Activity Ratio lysopc/pc lysopc synthesis Inflammation IDO-Activity Ratio Kyn/Trp Tryptophan breakdown Immune modulation 21

22 Case Study: Advantage of Broad Metabolite Coverage Collaboration: Prof. Burwinkel, Heidelberg University, Germany Component 2 (16.2%) Identification of key metabolites of ovarian cancer in plasma AA and AC are the top markers Importance of affected pathways in tumor pathophysiology and other diseases Component 1 (83.8%) Sample Description Sample Number Healthy Controls 15 Ovarian Cancer Patients 69 Unique metabolite portfolio of provides added value 22

23 Metabolic Signatures: Newborn Screening Beger et al., Metabolomics (216) 12: 149 Newborn screening Phenylketonuria (PKU) Phe, Phe/Tyr > 2.5 Medium-chain acyl-coa dehydrogenase (MCAD) deficiency C6 >.21, C8 >.32, C1 >.48, C1:1 >.28 C8/C1 > 1.6 or C8/C12 >

24 Metabolic Signatures: Precision Medicine Beger et al., Metabolomics (216) 12: 149 Val + Leu + Ile Phe + Tyr + Trp BCAA AAA Fischer ratio BCAA/AAA ratio with severity of liver failure Fischer et al., Am J Surg (1974) 127: 4 24

25 Summary & Conclusions The is a ready-to-use solution designed for targeted metabolomics on Q Exactive Orbitrap HRAM MS. Ready to go within a day, without method development The kit provides quantitative results for up to 48 metabolites covering a broad range of biological functions. The kit delivers a standardized, quality controlled, and targeted assay for accurate and confident metabolic profiling. The simple, efficient, and automated workflow ensures high throughput and use of minimal sample volume. The robust platform with standardized procedures assures longitudinal and inter-lab comparability. Standardized and Reproducible Quantitation of Hundreds of Lipids & Small Molecules for Broad Lipid and Metabolic Profiling 25

26 Acknowledgements BIOCRATES Pham Tuan Hai Therese Koal Doreen Kirchberg Martin Buratti Simon Schafferer Kristaps Klavins Barbara Wolf Brigitte Pfurtscheller Markus Langsdorf Manuel Kratzke Matthias Scheffler Anton Grones Wulf Fischer-Knuppertz Thermo Fisher Scientific Andreas Huhmer Anastasia Kalli Madalina Oppermann Claudio DeNardi David Peake Reiko Kiyonami Glenn Damkroeger Dirk Brouwer Neli Jochim Heather Sumner Klaus Lindpaintner 26

Thank you! Contact us for more information Helen.Karuso@biocrates.

27 Thank you! Contact us for more information BIOCRATES Life Sciences AG, Eduard-Bodem-Gasse 8, 62 Innsbruck, Austria 27 Not for use in diagnostic procedures.

28 Universal Selling Points of p4 HR How to transport the messages? Feature Standard Instrumentation Broad Installed base 48 endogenous metabolites from 11 metabolite classes lipids & 43 small molecules Complete Standardized & Quality controlled All-inone Solution transparent streamlined Workflow Manager MetIDQ guided process Automated Signal identification, -quantification and technical validation 96-well plate format, solid phase mediated sample preparation Quantitative Results with a High Accuracy, Intraday comparability & multi center + instrument reproducibility USP / Benefit No additional investments required Easy accessibility Unique broad Metabolic and Lipid Coverage gaining insights in to Cell Signaling & Structural Information + Bioenergetics (Acyl-carnitine, AS & Biogenic Amines) Ready to use & user friendly (No instrument tuning and method development) High confidence into analytical results High confidence into analytical results High-throughput, multiplexing, small sample volume Robust Platform necessary for Longitudinal & Interlab/study comparability BIOCRATES Life Sciences AG, Eduard-Bodem-Gasse 8, 62 Innsbruck, Austria 28

Metabolic Phenotyping Surrogate Marker for

Microbiota Lifestyle & Nutrition Environment Drug

Gender Age Systemic Analysis Acquired Metabolic

response - B cell response - T cell response

29 Metabolic Phenotyping Surrogate Marker for Individual Phenotype Exposure External pathogens Microbiota Lifestyle & Nutrition Environment Drug treatment Disposition Immune system Blood group Gender Age Systemic Analysis Acquired Metabolic homeostasis (compensation mechanism) Immune response - B cell response - T cell response Somatic Changes Genetic alteration Epigenetic regulation Transcriptional regulation 29

30 Targeted Metabolomics Overcoming Challenges of Untargeted Metabolomics Data NOT comparable NOT lab to lab, NOT study to study, NOT longitudinal Data Can Be Pooled lab to lab, study to study, longitudinal Fold Changes NOT standardized & NOT reproducible 8 samples Absolute Concentrations standardized & reproducible ~ 2 months 3 hours Signal Annotation (Database) 1 PhD scientist -4 Metabolites 1 Technician 4 Metabolites Biocrates Kits Software - Workflow Manager - Methods & Protocols - Signal Translation Peak Interpretation Reagents - Standards - Controls Unidentified Features Consumables - Kit Plate Instrument 1 Instrument 2 Instrument 1 Instrument 2 Untargeted Metabolomics Targeted Metabolomics 3

environmental influences on health (incl.

Understanding of Alzheimer's disease and

Nutritech: Effect of diet on phenotyping

31 Biocrates is Partner of Choice Large Scale, Long Term Studies Cardiology Lifestyle & Genomics Neurology SysVasc: HELIX: BrainAge: Identification of molecular targets for vascular disease treatment BiomarCaRE: Diabetes Biomarkers for cardiovascular risk assessment DeTecT2D: Development of a new diagnostic tool for early detection of type 2 diabetes Impact of early environmental influences on health (incl. 6 European birth cohorts) Nephrology SysKid: Understanding chronic kidney disease (diabetes and hypertension context) Impact of prenatal stress on brain ageing ADNI (1, GO, 2): Understanding of Alzheimer's disease and early diagnosis Nutrition & Lifestyle Nutritech: Effect of diet on phenotyping flexibility Epidemiological Cohorts KORA (F3, S4, F4) UK Twins EPIC CHRIS Leiden Longevity Study PopGen ERF Fenland Others BIOCRATES Life Sciences AG, Eduard-Bodem-Gasse 8, 62 Innsbruck, Austria 31

AbsoluteIDQ p180 Kit. Targeted Metabolite Identifi cation and Quantifi cation

AbsoluteIDQ p180 Kit. Targeted Metabolite Identifi cation and Quantifi cation AbsoluteIDQ p180 Kit Targeted Metabolite Identifi cation and Quantifi cation Bringing our targeted metabolomics expertise to your lab. The Biocrates AbsoluteIDQ p180 mass spectrometry Assay Preparation